Cytochrome c oxidase (COX) of the mitochondrial electron transport chain catalyzes the reduction of oxygen to water while concomitantly translocating protons across the inner mitochondrial membrane. This two part dissertation is a structural and functional investigation of COX using the bacterial model system from Rhodobacter sphaeroides (R.sph.).
First, the oligomeric structure of R.sph. COX within the lipid bilayer was investigated using discontinuous sucrose gradient ultracentrifugation. Utilizing this technique, liposomes containing R.sph. COX (pCOV) were separated from liposomes lacking enzyme (COV). The net buffering capacity and degree of light scattering per COX molecule were reduced in pCOVs, making them well suited for low buffer spectroscopic studies. Also, pCOVs maintained high oxygen reduction and proton pumping activities relative to COVs, indicating minimal damage induced by the centrifugation process. Quantitative lipid and protein concentrations were used to estimate the number of COX molecules per vesicle in the pCOVs. There was only one R.sph. COX molecule per vesicle, indicating that within the lipid bilayer, R.sph. COX exists in the monomeric state in contrast to the bovine enzyme which is dimeric. As a monomer, therefore, R.sph. COX is capable of maximal electron transfer and proton pumping efficiency.
Second, the structural and functional effects of a c-terminal subunit III truncation were characterized in R.sph. in order to gain insight into the critical role played by this subunit in proper COX functioning. The mutation was modeled after a human mitochondrial disease mutation which genetically truncates subunit III after the third of its seven helices (Delta114 COX). In R.sph. cells, Delta114 COX had lower expression levels and impeded rates of COX assembly. Altered levels of native in vivo processing of subunits II and IV were observed in Delta114 COX and in COX which had subunit III genetically removed (I-II COX). The truncated subunit III was incorporated into the COX complex with at least 70% stoichiometry and was subject to proteolytic processing at a specific cleavage site. Prior to enzymatic turnover induced inactivation, the proton pumping and oxygen reduction activities of Delta114 COX were half that of wildtype and equivalent to I-II COX at physiological pH. Delta114 and I-II COX had similar catalytic lifetimes in detergent micelle, but when supplemented with phospholipids from soybean, the catalytic lifetime of Delta114 COX was increased compared to I-II COX. Taken together, these results indicate that the c-terminal bundle of subunit III plays a role in the assembly of COX in R.sph. and in the native processing of subunits II and IV. They also highlight the role of the structural lipids within the v-shaped cleft of subunit III as being important for providing protection against turnover induced inactivation.